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Eukaryon

The Use of a Synthetic Transcription Activator in Activating the NIS Gene in the Presence of a Defective PAX8 gene.

Adrian Skobelski
Lake Forest College
Lake Forest, Illinois 60045

Abstract:       

            Mutations in the paired box gene 8 (PAX8), located predominantly in chromosome 2 in the human genome, have been linked to causing defects such as congenital hypothyroidism. Individuals who are born with a defective PAX8 gene resort to taking thyroid hormone medication for the rest of their lives to maintain adequate thyroid hormone levels. The need for developing genetic treatments to promote a normal developing thyroid from early embryogenesis has become an important area of interest. This paper presents a proposed experiment that will seek to expand our knowledge of designing artificial transcription factors through the use of zinc finger DNA binding domains that will recognize complementary sequences of enhancer binding regions, allowing for the continual expression of vital proteins during the presence of an inactive transcription factor. This experiment will seek to provide new insight into using artificial transcription factors as therapeutic strategies in treating congenital hypothyroidism during embryogenesis. Future studies on using this strategy on other genes such as the ones encoding thyroglobulin are necessary in order to assess the efficacy of this strategy.

 

PAX8: Leading Cause of Congenital Hypothyroidism

           

            The paired box gene 8 (PAX8) is an ideal example of how mutations in the sequence of a gene can result in defects in a variety of regions in the body. The PAX8 gene has been identified as a transcription factor that is commonly expressed in the developing kidneys, thyroid gland, and several areas of the central nervous system (“Paired Box Gene 8”, 1992). The role of a transcription factor is to control the rate of transcription by binding to a specific site on a DNA sequence, commonly referred to as the enhancer region. Mutations in the PAX8 gene have resulted in a variety of defects (phenotypes). These mutations have been classified primarily as loss of function mutations, which essentially means the mutations in the sequences of this gene lead to the gene products having few or no functions. Mutations can arise in a variety of ways; however, mutations in the PAX8 gene have been primarily identified as missense mutations, in which a change in one of the nucleotides (base pairs) have resulted in a different amino acid in the amino acid sequence of the gene protein product. The most common phenotypes resulting from mutations in the PAX8 gene include congenital hypothyroidism, thyroid dysgenesis, and irregular nephron differentiation during the development of the kidneys (“Paired Box Gene 8”, 1992).

            Congenital hypothyroidism, a condition in which there is an inadequate production of thyroid hormones in infants, occurs at a rate of 1 in every 3000-4000 births (“Congenital Hypothyroidism”, 2017). Multiple causes have been linked to the cause of this disease. Two potential causes of congenital hypothyroidism include thyroid dysgenesis and dyshormonogenesis (Ishizu et al. 2010). Thyroid dysgenesis can be defined as the condition in which the thyroid gland is completely missing (never developed) or in some cases, the thyroid gland is present, but it is not fully developed (Ishizu et al. 2010). Dyshormonogenesis is simply defined as the presence of inadequate thyroid hormone levels in an individual (Ishizu et al. 2010). Infants that are born with congenital hypothyroidism present symptoms such as decreased energy levels, poor growth, weight gain, and hoarse cry (“Congenital Hypothyroidism”, 2017). To be diagnosed, the babies will have to be tested for low levels of serum thyroid hormone (T4) and elevated levels of thyroid stimulating hormone (TSH) (“Congenital Hypothyroidism”, 2017). As a result of these fluctuating thyroid hormone levels, infants with congenital hypothyroidism will be subjected to taking thyroid hormone medication for many years in order to ensure stable thyroid hormone levels in the individual.

            Through a variety of studies, researchers have been able to locate and identify the locations of these mutations. The common tactic used to locate the site of the mutation is comparing the PAX8 sequences between family members who both demonstrate the phenotype. Two previous studies have identified heterozygous mutations in the PAX8 sequences, both resulting in loss-of-function mutations. One mutation was located to be at codon 55, which resulted in a substitution from histidine to glutamine, preventing the transcription of vital thyroid proteins, leading to thyroid dysgenesis (Palma et al. 2010). In another study, the autosomal dominant transmission of congenital hypothyroidism with thyroid hypoplasia was found to have been caused by a substitution of tyrosine to cysteine in codon 57 (Vilain et al. 2001). Further identification of PAX8 mutations is ideal in attempting to potentially use the recently developed CRISPR system to repair the mutations as one way of treating congenital hypothyroidism.

           

Molecular Function of the PAX8 Gene

 

PAX8 and Thyroid Development

           

            The PAX8 gene, a transcription factor, regulates the expression of several genes that encode important proteins including thyroglobulin, thyroid peroxidase, and sodium-iodide symporter. All three of these proteins are critical for a normal functioning thyroid gland, in that all three contribute to the production of important thyroid hormones known as T4 and T3 (Magliano et al. 2000). In a previous study, a group of researchers completed their study of observing the role of PAX8 in thyroid differentiation in early development. In the study, the researchers transformed rat thyroid cells with a polyoma virus middle T antigen in order to decrease the expression of the thyroglobulin, thyroid peroxidase, and sodium-iodide symporter (Magliano et al. 2000). The decrease in the expression of these proteins were connected to the loss of the PAX8. Reintroduction of PAX8 into cells that lacked PAX8 resulted in an increase of expression levels of the thyroglobulin, thyroid peroxidase, and sodium-iodide symporter, thus supporting the idea that PAX8 is necessary to activate transcription of thyroid-specific genes (Magliano et al. 2000). Understanding how the PAX8 gene results in the transcription of these thyroid-specific genes became more understood in another study, in which the researchers discovered the role of PAX8 and its binding capabilities to thyroid hormone enhancers. The researchers were able to identify the binding of the PAX8 to the enhancer region of the sodium-iodide symporter gene (NIS) (Ohno et al. 1999). It was found that the PAX8 would bind to two sites on the enhancer region NUE, which is responsible for the transcription of the sodium-iodide symporter (Ohno et al. 1999). In addition, they successfully identified mutations that affect the binding between the PAX8 and the enhancer region, which resulted in decreased transcription of NUE, thus a decrease in the expression of the sodium-iodide symporter (Ohno et al. 1999). These studies demonstrated the importance of the PAX8 gene in the transcription process of important thyroid hormones.

            In addition, previous studies have demonstrated the impactful role that the PAX8 gene on early thyroid development. Through the use of techniques such as in situ hybridization and immunohistochemistry, researchers have been able to visualize the expression of the PAX8 gene during early stages of embryonic development. In one previous study, the use of probes complementary to the PAX8 sequence, demonstrated the expression of the PAX8 gene in the median thyroid primordium and the thyroglossal duct during development (Trueba et al. 2005). The visualization of PAX8 expression in the developing thyroid supports the idea that the gene is vital to proper development of the thyroid, starting from embryogenesis.

 

The Use of a Mouse Model

 

            The use of mice as model organisms have been constantly used in research studies due to the vast number of genes that humans and mice share. When using knock-out mice, researchers “knockout” or inactivate a certain gene in the mice DNA and study the phenotypic changes that arise as a result of disrupting that gene. One example in which the use of knock-out mice was essential in studying the effects of an inactive PAX8 gene was in a study from 2003, in which the researchers studied the serum levels of important thyroid hormones T4 and T3. In PAX8-/-(inactive PAX8), the serum levels of T4 and T3 in these mice remained below detection limits, while control mice demonstrated maximum levels of T4 and T3 two weeks into the study (Friedrichsen et al. 2003). The use of knock-out mice in this study allowed researchers to discern the phenotypes that are expressed by active PAX8 genes and inactivated ones due to a possible mutation.

 

Experiment for the Future:

 

            In order to further the molecular genetics research in the field of congenital hypothyroidism, the ideal experiment would focus on finding ways to maintain proper thyroid hormone levels, but more importantly, to maintain stable expressions of the important thyroid proteins. Currently, patients who are being treated for congenital hypothyroidism must take thyroid hormones to maintain stable thyroid hormone levels, such as T4 and T3. As identified through previous research studies, inactivation of the PAX8 gene due to loss-of-function mutations, leads to the manifestation of improper thyroid development. Without the ability of the PAX8 gene to bind to the enhancer regions of genes that express proteins such as the sodium iodide symporter, the thyroid cells will lack important proteins to maintain regular functions. Recent advances in genetic engineering, such as the use of clustered regularly interspaced palindromic repeat (CRISPR) models and gene therapy, provide a glimpse into new therapeutic treatments being developed. One fairly recent development, the use of synthetic transcription factors, will be the primary focus in this proposal.

            This paper will be based on methods completed by previous studies. In a recent study from 2014, a group of researchers used the cre recombinase system to inactivate the PAX8 gene in healthy adult mice and observe the effects of an inactivated PAX8 gene in adults on the thyroid gland, unlike previous studies that focused on an inactivated PAX8 in developing mice. To inactivate the PAX8 gene, cre recombinase can be used in order to delete specific exons of the gene. Cre recombinase, an enzyme derived from the P1 bacteriophage, has been used in genetic studies to carry out mutagenesis of transgenes (Van Duyne 2015). This enzyme can result in the following: gene inversion, deletion, and translocation. All three of those events lead to an inactive gene of choice. To complete an experiment, two 34-bp recombination sites (LoxP sites), are selected in order to complete one of the three tasks on the sequence in between the sites. To express the cre recombinase, one may insert the gene for cre recombinase into a cell using a virus or one can create transgenic mice for which the gene for cre recombinase can be inserted in front of a promoter (Van Duyne 2015). For this experiment, a similar approach will be taken as seen in a previous study, in which the use of cre recombinase to delete two exons from the PAX8 gene sequence. To develop the necessary artificial transcription factors, methods from a previous study will also be incorporated. In this study, artificial transcription factors were designed to activate the human erythropoietin gene. The designed transcription factors were successful in activating transfected DNA templates and provided further insight on the importance of DNA binding affinity during the activation of a particular site (Zhang et al. 2000). Designing these artificial transcription factors to bind to a complementary enhancer region to promote the expression of a thyroid protein will be the primary focus in this proposed experiment.

 

Specific Aims

 

            As previously outlined, studies have demonstrated that the lack of a functional PAX8 gene leads to the inability to increase expression of important thyroid proteins. The primary focus of this experiment will focus on the binding relationship between the PAX8 gene and the enhancer region of the sodium iodide symporter gene. This proposed experiment will seek to further advance the study on artificial transcription factors, as a mean of continually expressing a protein despite the inactivation of the usual transcription factor (PAX8). The hypothesis of this study would be that the addition of a formulated artificial transcription factor, containing the complementary sequence of the enhancer region that is upstream of the sodium iodide importer, to transgenic mice containing an inactive PAX8 gene, will result in an increased expression of the sodium iodide importer protein.

 

Experimental Proposal

 

            To begin, a bacterial chromosome taken from mouse DNA will be manipulated to obtain a vector that contains loxP sites in the proper locations. The placement of the loxP sites, which are selected to knock out exons 3 and 4 from the wild-type allele, will be located near intron 2 and intron 4 (Marrota et al. 2014). Introns do not code for proteins and thus they are usually spliced out during the maturation of mRNA, while exons are the important sequences that encode for the expression of proteins. With the second loxP site, a neomycin-resistant cassette and a flip recombinase target (FRT) (Marrota et al. 2014). Both loxP sites will be oriented in the same direction, in order to delete the two PAX8 exons. With two PAX8 exons deleted, the PAX8 protein will be rendered useless, and in theory, mimicking a loss-of-function mutation. The vector will then have to be electroporated into a culture of embryonic stem cells (Marrota et al. 2014). To ensure proper electroporation of the modified vector, the embryonic stem cells will be placed in a medium consisting of neomycin, to which non-transfected cells will be killed off due to the missing neomycin cassette. Following the removal of the cassette, a clone of the embryonic stem cells will be inserted into a blastocyst (later forms into an embryo), generating heterozygous mice that contain an allele with the loxP sites (PAX8fl/+) (Marrota et al. 2014). These mice are then bred with donated PAX8Cre/+ mice, which are mice that contain the cre recombinase (Marrota et al. 2014). The cross breed between both mice types leads to the production of mice that include a variety of genotypes, but the most important of all being PAX8Cre/fl or mice that contain both the cre recombinase and the manipulated allele with the loxP sites (Marrota et al. 2014). With the cre recombinase being expressed, and the loxP sites being arranged in the same direction on the manipulated allele, the cre recombinase is able to delete the PAX8 exons.

            When a litter of PAX8Cre/fl mice are collected, the next step will be to construct an artificial transcription factor that would bind to the identified enhancer region of the NIS gene that expresses the sodium iodide symporter protein. Previous studies have identified the significant enhancer sequence binding site that the PAX8 binds to in order to increase the expression of the NIS gene. The identified enhancer region of the NIS gene that the PAX8 gene binds to has been previously located between nucleotides -2264 and -2495 in the 5’ flanking region of the sodium iodide symporter gene (Ohno et al. 1999). 232 novel zinc finger binding domains will be constructed in order to recognize the 231 bp sequence in the 5’ flank of the NIS gene (Zhang et al. 2000). These zinc finger domains will then be linked to the VP16 transcriptional activation domain, which will allow for the expression of the intended NIS gene (Zhang et al. 2000). FRTL-5 cells, also regarded to as the rat thyroid cells, will be extracted from the PAX8Cre/fl mice, to which the newly constructed synthetic transcription factors will be transfected to. The rat thyroid cells will be introduced back into the mice (Zhang et al. 2000). Immunohistochemical staining will be used to observe the expression of the sodium iodide symporter of three groups of mice: control group (PAX8+/+), mice with just the inactive PAX8 (PAX8Cre/fl), and the PAX8Cre/fl mice with the synthetic transcription factor (PAX8Cre/fl + 232 zinc finger domain).

            If the experiment goes as proposed and if there are increased expression levels of the NIS gene, then it would be reasonable to interpret that that synthetic transcription factors can be used when regular transcription factors are inactive. If there are similar levels of NIS expression as compared to the PAX8Cre/fl mice, then it can be concluded that this specific enhancer for the NIS gene only binds to the PAX8 gene. The greatest potential pitfall of this experiment will be designing the synthetic transcription activator, seeing as to how this method has recently became more frequently studied.

 

Conclusion:

            In conclusion, previous studies have demonstrated an incredible role of the PAX8 gene during early thyroid development. Mutations to the gene have been linked to causing congenital hypothyroidism, a condition in which children develop growth problems and weight gain due to the insufficient production of thyroid hormones such as thyroxine. Outlined in this proposal was an experiment in which the formation of synthetic transcription factors complimentary to an enhancer region of a gene that expresses the sodium iodide symporter, will be used on transgenic mice that contain an inactive PAX8 gene through the use of cre recombinase, in order to further the study of artificial transcription factors as possible therapeutic treatment options for infants diagnosed with congenital hypothyroidism.

 

References:

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Appendix A:

Skobelski Fig 1

Figure 1: Diagram representation of inactivating the PAX8 gene via cre recombinase. LoxP sites were designed facing the same direction in order to knockout two exons from the PAX8 gene sequence. The inclusion of a neomycin resistance cassette was inserted in order to ensure proper transfection of the targeting vector. The recombined allele represented an inactive PAX8 gene. (Marrota et al. 2014).

 

Skobelski Fig 2

Figure 2: Representation of designing a single zinc finger domain. (A) Visual of how the zinc finger domain interacts with the DNA alpha α- helix and β-sheet regions. (B) Design of amplifying the oligonucleotides that will recognize the complementary enhancer sequence region. (C) Depiction of the zinc finger protein created. (Zhange et al. 2000)

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